How are the cells on the surface of embryoid body derived from ES cells induced to differentiate into primitive endoderm?

在本研究项目中，我们首先假设来源于整合素与细胞外基质的粘附和钙粘蛋白的细胞间粘附的细胞外信号与可溶性因子协同参与小鼠ES细胞多能性的维持。为了测试该模型，我们绘制了通过过度表达整合素连接激酶（ILK）的显性负突变体抑制整联蛋白信号，并发现它在分化抑制剂LIF的存在下部分诱导分化。我们还尝试在悬浮培养条件下测试E-钙粘蛋白介导的信号在诱导聚集体表面向原始内胚层分化方面的功能。当我们将E-cadherin胞外区的可溶性形式放入培养物中时，原始内胚层的分化受到干扰，这表明E-cadherin信号量的减少可能触发位于聚集体表面的细胞的分化，这些胞外信号通过调节转录因子的表达来决定细胞的命运。我们发现锌指转录因子Gata 6的异位表达诱导ES细胞分化为原始内胚层细胞，其模仿胚胎来源的XEN细胞的表型。作为通过转录因子控制细胞命运的模型系统，我们揭示了决定滋养外胚层分化的Cdx 2和多能性相关转录因子Oct 3/4之间的相互抑制系统涉及多能细胞和滋养外胚层谱系之间的分离。此外，我们还阐明了另一个多能性相关转录因子Sox 2在ES细胞中的作用机制。这些发现将有助于分析指导原始内胚层分化的机制。

In this research project, we first hypothesized that the extracellular signals derived from the adhesion to the extracellular matrix via integrin and the cell-to-cell adhesion via cadherin would involve in the maintenance of pluripotency in mouse ES cells in cooperation to the soluble factors. To test this model, we plotted the inhibition of the integrain signal by overexpressing the dominant-negative mutant of integrin-linked kinase (ILK) and found that it partially induce differentiation in the presence of differentiation inhibitor LIF. We also tried to test the function of the E-cadherin-mediated signal on induction of differentiation toward primitive endoderm on the surface of aggregates in suspension culture condition. When we put the soluble form of the extracellular domain of E-cadherin in this culture, differentiation of primitive endoderm is interfered, suggesting that the reduced amount of E-cadherin signal might trigger differentiation in the cells locating the surface of the aggregates.These extracellular signals determine the cell fate by modulating the expression of the transcription factors. We found that the ectopic expression of zinc-finger transcription factor Gata6 induces differentiation of ES cells into primitive endoderm cells that mimic the phenotype of embryo-derived XEN cells. As the model system to control cell fate by transcription factors, we revealed that the reciprocal inhibition system between Cdx2, which determines differentiation to trophectoderm, and the pluripotency-associated transcription factor Oct3/4 involves in segregation between pluripotent cell and trophectoderm lineages. In addition, we also made it clear how the other pluripotency-associated transcription factor Sox2 works in ES cells. These findings will contribute to analyze the mechanism to direct primitive endoderm differentiation.